Early IGF-1 receptor inhibition in mice mimics preterm human brain disorders and reveals a therapeutic target

Besides recent advances in neonatal care, preterm newborns still develop sex-biased behavioral alterations. Preterms fail to receive placental insulin-like growth factor-1 (IGF-1), a major fetal growth hormone in utero, and low IGF-1 serum levels correlate with preterm poor neurodevelopmental outcomes. Here, we mimicked IGF-1 deficiency of preterm newborns in mice by perinatal administration of an IGF-1 receptor antagonist. This resulted in sex-biased brain microstructural, functional, and behavioral alterations, resembling those of ex-preterm children, which we characterized performing parallel mouse/human behavioral tests. Pharmacological enhancement of GABAergic tonic inhibition by the U.S. Food and Drug Administration–approved drug ganaxolone rescued functional/behavioral alterations in mice. Establishing an unprecedented mouse model of prematurity, our work dissects the mechanisms at the core of abnormal behaviors and identifies a readily translatable therapeutic strategy for preterm brain disorders.


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Figs. S1 to S13 Tables S1 to S18 C T R L      Table S10.GNX treatment (see Fig. 5) does not improve myelination deficit in JB1-treated adolescent mice.

Figure S2 .Figure S3 .Figure S5 .Figure S7 .Figure S8 .Figure S9 .
Figure S2.Systemic IGF-1R inhibition in WT mouse pups leads to acute phospho-proteomic changes associated with neuron and synapse development (A) Experimental protocol with pharmacological treatment and timing of the phospho-proteomic experiment.(B) Heatmap showing clustering of significant (FDR <0.05) dephosphorylated (cluster 1) or hyperphosphorylated (cluster 2) phosphosites in JB1-treated pups sacrificed at the end of the treatment.Values are normalized on z-score.(C) Gene Ontology (GO, cellular compartment, and molecular function) and Reactome pathway analysis for the significantly differentially dephosphorylated (left) or hyperphosphorylated (right) proteins (from the same analysis in Figure1B).The color bar on the right indicates -log10FDR for the statistically significant (FDR <0.05) top 20 (hierarchy for fold enrichment) enriched cellular compartments, molecular functions or Reactome pathways.Terms related to neurons or synapses are underlined in red.Schematic cartoons by BioRender.com.

Figure S10 .F
Figure S10.Early postnatal IGF-1R inhibition leads to a significant regional-and sex-biased loss of interneurons in adolescent mice.(A) Experimental protocol with pharmacological treatment and timing of histological experiments.(B) Quantification of the density of PV interneurons in the same experiments as in Fig. 3G, with data segregated by animal's gender.Bars represent the average density of PV-positive cells of all the analysed animals' ± SEM, and symbols represent single data points for each animal.Left, two-tailed Student's t-test, t = 3.099, *p < 0.05.(C) Quantification of the density of SST interneurons in the same experiments as in Fig. 3K, with data segregated by animal's gender.Bars represent the average density of SST-positive cells of all the analysed animals ± SEM, and symbols represent single data points for each animal.(D) Quantification of the density of PV interneurons in the same experiments as in Fig. 3I, with data segregated by animal's gender.Middle left, two-tailed Student's t-test, t = 2.494, *p < 0.05.Bars represent the average density of PV-positive cells of all the analysed animals ± SEM, and symbols represent single data points for each animal.*p<0.05,Student's t test.(E) Quantification of the density of SST interneurons in the same experiments as in Fig. 3M, with data segregated by animal's gender.Bars represent the average density of SST-positive cells of all the analysed animals ± SEM, and symbols represent single data points for each animal.Left, two-tailed Student's t-test, t = 3.906, *p<0.05.Schematic cartoons by BioRender.com.

Figure S12 .Figure S13 .
Figure S12.Preterm children without diagnosed neonatal brain injury perform below standard norms in cognitive tests and show increased autistic traits.(A) Timeline for behavioural evaluation of ex-preterm children of the same cohort presented in Fig.S11.(B) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the VSI test.The dashed line represents standard norm mean of typically developing children from the literature.One-sample t-test, t = 5.805, ***p < 0.0001.(C) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the PSI test.The dashed line represents standard norm mean of typically developing children from the literature.Onesample t-test, t = 4.305, ***p < 0.0001.(D) Quantification of the mean ± SEM (overlying line) and single expreterm child scoring (symbols) of the WMI test.The dashed line represents standard norm mean of typically developing children from the literature.One-sample t-test, t = 8.883, ***p < 0.0001.(E) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the VSI test presented in B, with data segregated for gender.The dashed line represents standard norm mean of typically developing children from the literature.Left, one-sample t-test, t = 3.570, *p < 0.01.Right, one-sample t-test, t = 4.994, ***p < 0.0001.(F) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the PSI test presented in C, with data segregated for gender.The dashed line represents standard norm mean of typically developing children from the literature.Left, one-sample t-test, t = 3.220, *p < 0.01.Right, one-sample t-test, t = 3.478, *p < 0.01.(G) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the WMI test presented in D, with data segregated for gender.The dashed line represents standard norm mean of typically developing children from the literature.Left, onesample t-test, t = 4.946, ***p < 0.0001.Right, one-sample t-test, t = 8.114, ***p < 0.0001.(H) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the Zoo Locations test presented in Fig. S11D, with data segregated for gender.The dashed line represents standard norm mean.Left, one-sample t-test, t = 6.240, ***p < 0.0001.Right, one-sample t-test, t = 8.603, ***p < 0.0001.(I) Quantification of the mean ± SEM (overlying line) and single child ex-preterm scoring (symbols) of the Autism Quotient parent questionnaire presented in Fig. S11F, with data segregated for sex.The dashed line represents standard norm mean of typically developing children from the literature.Left, one-sample t-test, t = 5.458, ***p < 0.0001.Right, one-sample t-test, t = 4.254, **p < 0.001.(J) Quantification of the percentage number of ex-PT children with pathological scoring in the VSI test presented in B. The dashed line represents Percentile Ranks of standard norm of typically developing children from the literature with a cut off for scoring < 70.(K) Quantification of the percentage number of ex-preterm children with pathological scoring in the PSI test presented in C. The dashed line represent Percentile Ranks of standard norm of typically developing children from the literature with a cut off for scoring < 70.(L) Quantification of the percentage number of ex-preterm children with pathological scoring in the WMI test presented in D. The dashed line represents Percentile Ranks of standard norm of typically developing children from the literature with a cut off for scoring < 70.Fisher's exact test, ***p < 0.01; male vs female comparison, Fisher's exact test, # p < 0.05.Schematic cartoons by BioRender.com.
Table showing the parallelism between the behavioural tests performed in mice and in humans.(D)Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the zoo locations test.The dashed line represents the standard norm of typically developing children from the literature.One-sample t-test, t = 10.09,***p < 0.0001.(E) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the memory design delayed test.The dashed line represents the standard norm of typically developing children from the literature.One-sample t-test, t = 5.596, ***p < 0.0001.(F) Quantification of the mean ± SEM (overlying line) and single ex-preterm child scoring (symbols) of the Autism Quotient parent questionnaire.The dashed line represents the standard norm of typically developing children from the literature.One-sample t-test, t = 5.898, ***p < 0.0001.(G) Quantification of the percentage number of expreterm children with pathological scoring in the zoo locations test presented in D (males: 20 children; females: 31 children).The dashed line represents the percentile ranks of the standard norm of typically developing children from the literature with a cut off for scoring ≤ 6. Fisher's exact test, ***p < 0.0001.Male vs. female comparison, Fisher's exact test, #p < 0.05.(H) Quantification of the percentage number of expreterm children with pathological scoring in the memory design delayed (content score, left; spatial score, right) test presented in E (males: 20 children; females: 31 children).The dashed line represents percentile ranks of the standard norm of typically developing children from the literature with a cut off for scoring ≤ 5th percentile rank.Fisher's exact test, ***p < 0.0001.(I) Quantification of the percentage number of ex-preterm children scoring above the autism cut off in the Autism Quotient parent questionnaire presented in F (males: 20 children; females: 31 children).The dashed line represents the percentage of children in the standard norm of typically developing children from the literature with a cut off score ≥76.Fisher's exact test, *p<0.01,*** p<0.0001.Schematic cartoons by BioRender.com.

Table S2 . USV call classification for data presented in the experiments reported in
Fig.2B,C.Early IGF-1R inhibition leads to increased chevron-type calls in P6 pups.Two-tailed Student's t-test, t = 2.345, *p<0.05.

Table S1 .
Systemic IGF-1R inhibition in WT mouse pups does not induce any significant acute change in IGF-1 hippocampal and plasma levels and in IGF-1-related proteins plasma levels.LCMS: Liquid Chromatography-Mass Spectrometry

Table S5 .
Dephosphorylated -phosphorylation sites differentially expressed between JB1-treated (N = 5 animals) and vehicle-treated (control; N = 4 animals) female pup littermates sacrificed 1 hour after the last treatment at P5.Note that, for this analysis, we used S0=0.1 and P value<0.01.

Table S7 .
Kinase Enrichment Analysis of significantly dephosphorylated (left) and hyperphosphorylated (right) phospho-proteins corresponding to differentially expressed phosphorylation sites showed in TableS5,6.The kinases related to IGF-1 signalling are highlighted in bold.

Table S8 .
Enrichment for neuropsychiatric disorder risk-genes (identified with SFARI gene archive) in the dephosphorylated and hyperphosphorylated dataset shown in TableS5,6.

Table S9 .
Gene Ontology (GO) analysis for the differentially dephosphorylated or hyperphosphorylated significantly expressed proteins corresponding to differentially expressed phosphorylation sites showed in TableS5,6.

Table S18
. Neurodevelopmental and cognitive quotients of the investigated cohort of ex-premature children presented in Fig. S11 and Fig. S12.